Attenuated recombinant vesicular stomatitis virus vaccine vectors comprising modified matrix proteins

ABSTRACT

The present invention relates to vesicular stomatitis virus (VSV) matrix (M) protein mutants. One mutant M protein includes a glycine changed to a glutamic acid at position 21, a leucine changed to a phenylalanine at position 111 and a methionine changed to an arginine at position 51. Another M protein mutant includes a glycine changed to a glutamic acid at position 22 and a methionine changed to an arginine at positions 48 and 51. Yet another VSV M protein mutant includes a glycine changed to a glutamic acid at position 22, a leucine changed to a phenylalanine at position 110 and a methionine changed to an arginine at positions 48 and 51. The present invention is directed also to recombinant VSVs (rVSV) having these M mutants and to vaccines based on the rVSV having the M mutants of the present invention. These new rVSVs having the mutant M were significantly attenuated and lost virulence, including neurovirulence, and are capable of inducing an immune responses against an antigen of interest. In addition, a rVSV serotype Indiana having the first described M mutant is capable of efficient replication at 31° C., and of poor replication or incapable of replication at about 37° C. or higher.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/368,102 filed Jun. 23, 2014, which is a national stage application under 35 U.S.C. 371 of International Application No. PCT/CA2012/001185, filed Dec. 21, 2012, which in turn claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Ser. No. 61/579,902, filed Dec. 23, 2011, the contents of each of which are hereby incorporated by reference into the present disclosure.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

A paper copy of the Sequence Listing and a Sequence Listing in computer readable form in .txt format titled “118494_144SequenceListing.txt”, which was submitted online on Aug. 30, 2016, and is 22 KB in size are hereby incorporated by reference. Applicants assert that the paper copy of the Sequence Listing is identical to the Sequence Listing in computer readable form.

FIELD OF THE INVENTION

The present invention relates, in general, to biotechnology and immunology. In particular, the present invention relates to novel matrix (M) proteins of vesicular stomatitis virus (VSV), to attenuated recombinant VSVs expressing the novel M proteins and to VSV based prime-boost vaccines that induce long-lasting humoral, cell-mediated and mucosal immune responses against foreign antigens expressed by the recombinant VSV carrying foreign genes.

BACKGROUND OF THE INVENTION

Vesicular stomatitis virus (VSV) is a negative stranded RNA virus which infects most mammalian cells and expresses viral proteins up to 60% of total proteins in infected cells [Kim, G. N., and C. Y. Kang. Virology 357:41, 2007]. In nature VSV infects pigs, cattle, and horses, and causes vesicular disease around the mouth and foot. Although human infection by VSV has been reported, VSV does not cause any serious symptoms in humans [Fields, B. N., and K. Hawkins. N Engl J Med 277:989, 1967; Johnson, K. M. et al. Am J Trop Med Hyg 15:244, 1966].

VSV encodes five proteins, nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G), and RNA dependent RNA polymerase (L). The N, P, and L proteins of VSV are required for synthesis of positive sense and negative sense genomic RNAs and mRNA, which are necessary for the synthesis of VSV proteins.

Blocking the host cellular protein synthesis by VSV matrix (M) protein induces cell death. Changing a methionine residue at position 51 of the M protein to arginine (M51R) in the vesicular stomatitis virus Indiana serotype (VSV_(Ind)), and changing methionines at position 48 (Met48) and 51 (Met51) to Arg in the vesicular stomatitis virus New Jersey serotype (VSV_(NJ)) M gene could negate this function of VSV M protein (Kim, G. and Kang, C., Virology, 357:41-53, 2007). While the VSVs with these Met to Arg mutations in the M protein have significantly reduced cytopathic effects, they still replicate and produce progeny viruses in the cell and in the infected animal. One of the temperature sensitive (ts) M gene mutants of VSV_(Ind) Orsay strain, tsO23 has shown limited replication in a mouse glial cell line at 37° C. and 39° C. (Rabinowitz, S. et al. Infection and Immunity 33:120-125, 1981). It has been demonstrated that the temperature sensitivity of tsO23 was the result of the improper or lack of the initiation of the viral assembly with the characteristic bullet shaped structure at 39° C. (Flood, E. et al. Virology 278:520-533, 2000; Lyles, D. et al. Virology, 217:76-87, 1996). In addition, the tsO23 lost its neurovirulence in mice even after direct inoculation into the brain (Rabinowitz, S. et al. Infection and Immunity, 33:120-125, 1981).

There are three amino acid differences between wild type M of VSV Orsay strain and M of tsO23, which are glycin (G) to glutamic acid (E) at 21st amino acid (G21E), leucine (L) to phenylalanine (F) at 111th amino acid (L111F), and histidine (H) to tyrosine (Y) at 227th amino acids (H227Y) (Morita, K. et al. J. Virol. 61:256-263, 1987). The single amino acid reversion at F111 to L in revertants implicated that the F111 appears to play a major role in the temperature sensitivity of the tsO23. However, the other two mutations may also have some role because the single reversion from the F to L at 111th position did not recover the virus completely to its wild type phenotype (Morita, K. et al. J. Virol. 61:256-263, 1987; Li, Y. et al. J. Virol. 62:3729-3737, 1988).

Currently, research groups have developed replication competent, assembly defective VSV having a G glycoprotein (G) gene deleted (AG) or both G and M genes deleted (AMG) as safer vaccine vectors (Kahn, J. et al. J. Virol. 75:11079-11087, 2001; Schwartz, J. et al., Virology 366:166-73, 2007). However, having the G gene deleted or M and G gene deleted, the VSV vector requires the supply of G or both M and G proteins in trans for the production of the assembly-defective VSV. In order to reduce the cost of producing vaccines, it is necessary to generate a system, which can produce the viral vaccine vectors that can replicate in high titre. Therefore, what is needed is a full-length VSV vector system, which has lost its virulence (avirulent) and still can replicate to a high titer in vitro at 31° C. cannot assemble properly at non-permissible temperatures, and induce good immune responses against the gene of interest that it expresses.

Further and other objects of the invention will be realized from the following Summary of the Invention, the Discussion of the Invention and the embodiments and Examples thereof.

SUMMARY OF THE INVENTION

The inventors have generated new matrix (M) protein mutants of vesicular stomatitis virus (VSV), including VSV Indiana serotype (VSV_(Ind)) and VSV New Jersey serotype (VSV_(NJ)). These new VSVs having the mutant M proteins are essentially non-cytolytic, significantly attenuated and avirulent, including neurovirulence (safer). Furthermore, these new VSVs having the mutant M proteins are capable of inducing immune responses against an antigen or epitope of interest. The immune response may be humoral, cellular and mucosal immune responses. In addition, the VSV_(Ind) having the M mutant can assemble and be released from the infected cells normally at 31° C., but they can not assemble properly at 37° C. or higher and the release of the viruses from infected cells is reduced significantly by about 1000 or 10,000 folds compared to that of wild type VSV_(Ind).

As such, in one embodiment, the present application relates to a modified matrix (M) protein of a vesicular stomatitis virus (VSV). In one embodiment, the modified M protein comprises an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R; and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R. In one aspect of the present invention, the modified M protein of the present invention comprises the amino acid sequence of SEQ ID NO:8 including at least the following substitutions: G22E/L110F/M48R+M51R.

In another embodiment of the modified M protein of the present invention, the modified M protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:10.

In another embodiment, the present application provides for a recombinant VSV (rVSV). In one embodiment, the rVSV comprises a modified matrix (M) protein, the modified M protein comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R; and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R.

In one embodiment of the present invention, the rVSV is a chimeric rVSV that expresses a protein of a foreign pathogen, and wherein said chimeric rVSV is capable of inducing an immune response to said protein.

In another embodiment of the rVSV of the present invention, the pathogen is a viral, fungal, bacterial or parasitic pathogen.

In another embodiment of the rVSV of the present invention, the rVSV is essentially non-cytolytic and avirulent.

In one embodiment of the present invention, the rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSV_(Ind)), and the modified M protein comprises the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111F/M51R.

In one embodiment of the rVSV_(Ind) of the present invention, the modified M protein comprises the amino acid sequence of SEQ ID NO: 4.

In another embodiment of the rVSV_(Ind) of the present invention, the modified M protein is encoded by a gene comprising a nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the rVSV of the present invention, the rVSV_(Ind) of any of the previous embodiments is capable of producing VSV_(Ind) particles at permissible temperatures and incapable of producing the particles at non-permissible temperatures.

In another embodiment of the present invention, the rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ)), and the modified M protein comprises the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R.

In one embodiment of the rVSV_(NJ) of the present invention, the modified M protein is encoded by a gene having a nucleotide sequence of SEQ ID NO: 6.

In another embodiment of the rVSV_(NJ) of the present invention, the modified M protein comprises the amino acid sequence SEQ ID NO: 9.

In another embodiment of the rVSV_(NJ) of the present invention the modified M protein includes the following substitutions: G22E/L110F/M48R+M51R.

In another embodiment of the rVSV_(NJ) of the present invention, the modified M protein is encoded by a gene having a nucleotide sequence of SEQ ID NO: 7.

In another embodiment of the rVSV_(NJ) of the present invention, the modified M protein comprises the amino acid sequence SEQ ID NO: 10.

In one embodiment, the present invention provides for a vaccine. In one embodiment, the vaccine of the present invention comprises an effective amount of one or more attenuated rVSVs, the one or more attenuated rVSVs including a modified matrix (M) protein, the modified M protein comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R, and (ii) SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R.

In one embodiment of the vaccine of the present invention, the rVSV is a recombinant vesicular stomatitis virus Indiana serotype (rVSV_(Ind)), and the modified M protein comprises the amino acid sequence of SEQ ID NO: 3 including at least the following substitution: G21E/L111F/M51R.

In another embodiment of the vaccine of the present invention, the modified M protein of the rVSV_(Ind) comprises the amino acid sequence of SEQ ID NO: 4.

In another embodiment of the vaccine of the present invention, the modified M protein of the rVSV_(Ind) is encoded by a gene comprising a nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the vaccine of the present invention, the rVSV_(Ind) is capable of producing VSV_(Ind) particles at permissible temperatures and incapable of producing the particles at non-permissible temperatures.

In one embodiment of the vaccine of the previous invention, the rVSV is a recombinant vesicular stomatitis virus New Jersey serotype (rVSV_(NJ)), and the modified M protein comprises the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R.

In one embodiment of the vaccine of the present invention, the rVSV is rVSV_(NJ) and the modified M protein is encoded by a gene having a nucleotide sequence of SEQ ID NO: 6.

In another embodiment of the vaccine of the present invention, the rVSV is rVSV_(NJ) and the modified M protein comprises the amino acid sequence SEQ ID NO: 9.

In another embodiment of the vaccine of the present invention, the rVSV is rVSV_(NJ) and the modified M protein includes the following substitutions: G22E/L110F/M48R+M51R.

In another embodiment of the vaccine of the present invention, the rVSV is rVSV_(NJ) and the modified M protein is encoded by a gene having a nucleotide sequence of SEQ ID NO: 7.

In another embodiment of the vaccine of the present invention, the rVSV is rVSV_(NJ) and the modified M protein comprises the amino acid sequence SEQ ID NO: 10.

In another embodiment of the vaccine of the present invention, the rVSV according to any of the previous embodiments is a chimeric rVSV that expresses a protein of a foreign pathogen, and the chimeric rVSV is capable of inducing an immune response to said protein.

In another embodiment of the vaccine of the present invention, the rVSV comprises a mixture of attenuated chimeric rVSVs, wherein at least two chimeric rVSVs in the mixture express a different protein of the foreign pathogen.

In another embodiment of the vaccine of the present invention, the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

In another embodiment of the vaccine of the present invention, the pathogen is a lentivirus.

In another embodiment of the vaccine of the present invention, the lentivirus is a HIV and the epitope is a HIV protein.

In another embodiment of the present invention, the pathogen is HCV and the epitope is a HCV protein.

In one embodiment of the present invention, the vaccine of any one of the previous embodiments is capable of inducing a humoral, cellular and mucosal immune response.

In another embodiment of the present invention, the vaccine according to any one of the previous embodiments further includes an adjuvant.

In one embodiment, the present invention provides for a method of inducing an immune response in a subject. In one embodiment of the present invention, the method comprises administering to the subject: (a) an effective amount of a vaccine comprising an attenuated rVSV of one serotype having a first modified M protein, the first modified M protein comprising the amino acid sequence of SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R; and (b) an effective amount of another vaccine comprising an attenuated rVSV of another serotype having a second modified M protein, the second modified M protein comprising the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R. In one aspect of the present invention, the second modified M protein comprises the amino acid sequence of SEQ ID NO:8 including at least the following substitutions: G22E/L110F/M48R+M51R

In one embodiment of the method of the present invention, (a) is administered to the subject before (b) is administered to the patient.

In another embodiment of the method of the present invention, (b) is administered to the subject more than one time over the course of inducing.

In another embodiment of the method of the present invention, (a) is administered to the subject and (b) is administered to the subject at about weeks three, eight and sixteen post-administration of (a).

In another embodiment of the method of the present invention, (b) is administered to the subject before (a) is administered to the subject.

In another embodiment of the method of the present invention, (a) is administered to the subject more than one time over the course of inducing.

In another embodiment of the method of the present invention, (b) is administered to the subject and (a) is administered to the subject at about weeks three, eight and sixteen post-administration of (b).

In another embodiment of the method of the present invention, the rVSV of vaccine (a) and rVSV of vaccine (b) are chimeric rVSVs that express a protein of a foreign pathogen, and wherein the two rVSVs are capable of inducing an immune response to the protein.

In another embodiment of the method of the present invention, the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

In another embodiment of the method of the present invention, the pathogen is a lentivirus.

In another embodiment of the method of the present invention, the lentivirus is a HIV and the protein is a HIV protein.

In another embodiment of the method of the present invention, the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is a HIV gene inserted in between the G gene and the L gene.

In another embodiment of the method of the present invention, the HIV gene is selected from the group of HIV genes consisting of gag, env and pol.

In another embodiment of the method of the present invention, the pathogen is HCV and the protein is a HCV protein.

In another embodiment of the method of the present invention, the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene of the rVSV.

In another embodiment of the method of the present invention, vaccine (a) and vaccine (b) comprise a mixture of attenuated chimeric rVSVs, wherein at least two of the attenuated chimeric rVSVs in the mixture express a different protein of the pathogen.

In another embodiment of the method of the present invention, each one of the two vaccines (a) and (b) induces humoral, cellular and mucosal immune responses.

In another embodiment of the method of the present invention, the serotype of the rVSV of vaccine (a) is Indiana and the serotype of the rVSV of vaccine (b) is New Jersey.

In another embodiment of the method of the present invention, each of vaccine (a) and vaccine (b) further comprises an adjuvant.

In one embodiment, the present invention provides for a prime boost combination vaccine. In one embodiment, the prime boost combination vaccine comprises: (a) an effective amount of a vaccine comprising an attenuated rVSV of one serotype having a first modified M protein comprising the amino acid sequence of SEQ ID NO:4; and (b) an effective amount of a vaccine comprising a rVSV of another serotype having a second modified M protein comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

In one embodiment of the prime boost combination vaccine of the present invention, vaccine (a) is a priming vaccine and vaccine (b) is a booster vaccine.

In another embodiment of the prime boost combination vaccine of the present invention, vaccine (b) is a priming vaccine and vaccine (a) is a booster vaccine.

In another embodiment of the prime boost combination vaccine of the present invention, the two attenuated rVSVs are chimeric rVSVs that express a protein of a foreign pathogen, and the two chimeric rVSVs are capable of inducing an immune response to the protein.

In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.

In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is a lentivirus.

In another embodiment of the prime boost combination vaccine of the present invention, the lentivirus is a HIV and the protein is a HIV protein.

In another embodiment of the prime boost combination vaccine of the present invention, the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is inserted in between the G gene and the L gene.

In another embodiment of the prime boost combination vaccine of the present invention, the HIV gene is selected from the group of HIV genes consisting of env, gag and pol.

In another embodiment of the prime boost combination vaccine of the present invention, the pathogen is HCV and the epitope is a HCV protein.

In another embodiment of the prime boost combination vaccine of the present invention, the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene.

In another embodiment of the prime boost combination vaccine of the present invention, the HCV protein is a structural or a non-structural HCV protein.

In another embodiment of the prime boost combination vaccine of the present invention, each one of the two vaccines comprise a mixture of the attenuated chimeric rVSVs, and at least two of the attenuated chimeric rVSVs in the mixture have a different protein of the pathogen.

In another embodiment of the prime boost combination vaccine of the present invention, each one of the two vaccines is capable of inducing humoral, cellular and mucosal immune responses.

In another embodiment of the prime boost combination vaccine of the present invention, the serotype of vaccine (a) is Indiana and the serotype of vaccine (b) is New Jersey.

In another embodiment of the prime boost combination vaccine of the present invention, each one of vaccine (a) and vaccine (b) further comprises an adjuvant.

In one embodiment, the present invention provides for a kit comprising: (a) at least one dose of an effective amount of a vaccine comprising a rVSV_(Ind) having a modified M protein comprising the amino acid sequence of SEQ ID NO:4, and (b) at least one dose of an effective amount of a vaccine comprising rVSV_(NJ) having a modified M protein comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

In one embodiment of the kit of the present invention, (a) and (b) are formulated in a pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides for an isolated peptide comprising an amino acid sequence selected from the group of amino acid sequences listed as SEQ ID NOs: 4, 9 and 10.

In one embodiment, the present invention provides for isolated nucleotide sequences for encoding the isolated peptides of SEQ ID NOs: 4, 9 and 10.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments will now be described, by way of example only, with reference to the drawings, in which:

FIG. 1 illustrates gene organization of the vesicular stomatitis virus (VSV), Indiana (Ind) serotype with mutations in the M gene.

FIG. 2 illustrates gene organization of vesicular stomatitis virus (VSV), New Jersey (NJ) with mutations in the M gene.

FIG. 3 illustrates a reverse genetics system for the recovery of VSV from cDNA.

FIGS. 4A to 4D illustrate the burst size effect of mutation L111F in the M protein of rVSV_(Ind) at permissive temperature [FIG. 4A and FIG. 4C] and at semi-permissive temperature [FIG. 4B and FIG. 4D], in baby hamster kidney (BHK) cells infected with recombinant VSVs [FIG. 4A and FIG. 4B] and in human neuroblastoma cells (SH-SY5Y) infected with the rVSV.

FIG. 5 is a graph representing the burst size effect of mutations M48R+M51R, G22E, G22E/M48R+M51R, G22E/L110F, G22E/L110F/M48R+M51R in the M protein of rVSV_(NJ) at 31° C. and 37° C. in BHK21 cells.

FIG. 6A is a Western blot analysis illustrating the level of rVSV protein expression of rVSV_(Ind) 1. WT, 2. M51R, 3. G21E 4. G21E/M51R 5. G21E/L111F and 6. G21E/L111F/M51R.

FIG. 6B is a Western blot analysis illustrating the level of rVSV protein expression of rVSV_(NJ) 1. WT, 2. M48R+M51R, 3. G22E 4. G22E/M48R+M51R 5. G22E/L110F and 6. G22E/L110F/M48R+M51R.

FIGS. 7A to 7N are photographs of BHK21 cells infected with rVSV_(Ind) having a wild type M gene (FIGS. 7B and 7I) and with rVSV_(Ind) having different mutations in the M gene (FIGS. 7A, 7D, 7E, 7F, 7G, 7H, 7K, 7L, 7M and 7N) and photographs of uninfected BHK21 cells (FIGS. 7C and 7J). Cells were incubated at 31° C. (panels A to G) or 37° C. (FIGS. 7H to 7N).

FIGS. 8A to 8L are photographs of SH-SY5Y cells infected with rVSV_(Ind) having different mutations in the M gene (FIGS. 8C, 8D, 8E, 8F, 8I, 8J, 8K and 8L) or wild type M gene (FIGS. 8A and 8G) and photographs of uninfected SH-SY5S cells (FIGS. 8B and 8H). Cells were incubated at 31° C. (FIGS. 8A to 8F) or 37° C. (FIGS. 8G to 8L).

FIGS. 9A to 9N are photographs of BHK21 cells infected with rVSV_(NJ) having different mutations in the M gene (FIGS. 9A, 9D, 9E, 9F, 9G, 9H, 9K, 9L, 9M and 9N) or wild type M gene (FIGS. 9B and 9I), and photographs of uninfected BHK21 cells (FIGS. 9C and 9J). Cells were incubated at 31° C. (FIGS. 9A to 9G) or 37° C. (FIGS. 9H to 9N).

FIGS. 10A to 10N are photographs of SH-SY5Y cells infected with rVSV_(NJ) having different mutations in the M gene (FIGS. 10A, 10D, 10E, 10F, 10G, 10H, 10K, 10L, 10M and 10N) or having wild type M gene (FIGS. 10B and 10I), and photographs of uninfected SH-SY5Y cells (FIG. 10C and FIG. 10J). Cells were incubated at 31° C. (FIGS. 10A to 10G) or 37° C. (FIGS. 10H to 10N).

FIGS. 11A to 11 D are graphs illustrating neurovirulence studies in Swiss Webster mouse by intralateral ventricular injection: FIG. 11A UV-irradiated rVSV_(Ind) G21E/L111F/M51R, FIG. 11B rVSV_(Ind) WT, FIG. 11C rVSV_(Ind) M51R and FIG. 11D rVSV_(Ind) G21E/L111F/M51R.

FIGS. 12A to 12D are graphs illustrating neurovirulence studies in Swiss Webster mouse by intralateral ventricular injection: FIG. 12A rVSV_(NJ) WT, FIG. 12B rVSV_(NJ) M48R+M51R, FIG. 12C rVSV_(NJ) G22E/M48R+M51R and FIG. 12D rVSV_(NJ) G22E/L110F/M48R+M51R.

FIGS. 13A to 13C are graphs illustrating the generation of rVSVs expressing HIV-1 Gag protein as a gene of interest. FIG. 13A (SEQ ID NOS: 11-15) illustrates the cloning of HIV-1 Gag-En genes into the cDNA clone of rVSV. FIG. 13B is a Western blot analysis of BHK cells infected with rVSVs expressing Gag-En and having different mutations to the M gene, and incubated at 31° C. FIG. 13C is a Western blot analysis of BHK cells infected with rVSVs expressing Gag-En and having different mutations to the M gene, and incubated at 37° C.

FIG. 14 depicts a vaccination regime and a table of vaccination groups.

FIG. 15 depicts a graph of the frequency of peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with dimethyl sulfoxide (DMSO).

FIG. 16 is a graph illustrating the frequency of VSV N peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with VSV N.

FIG. 17 is a graph illustrating the frequency of HIV-1 Gag peptide specific CD8+ IFN gamma+ T cells among the different vaccination groups illustrated in the insert stimulated with HIV-1 Gag.

FIG. 18 is a graph illustrating HIV-1 Gag specific antibody responses among the different vaccination groups illustrated in the insert.

FIG. 19 is a graph illustrating the frequency of VSV N peptide specific CD8+ IFN gamma+ T cells among different vaccination groups vaccinated with various doses of rVSV of Table 1.

FIG. 20 is a graph illustrating the frequency of HIV-1 Gag peptide specific CD8+ IFN gamma+ T cells among different vaccination groups vaccinated with various doses of rVSV of Table 1.

FIG. 21 is a graph illustrating HIV-1 Gag specific antibody responses among different vaccination groups vaccinated with various doses of rVSV of Table 1.

FIG. 22 (SEQ ID NOS: 16-26) illustrates the cloning of HIV-1 gag gene linked to nucleotides encoding human B cell and T cell epitopes of gp120 and gp41 into the cDNA clone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ) G22E/M48R+M51R.

FIG. 23 (SEQ ID NOS: 11-15 and 27-31, respectively) illustrates the cloning of HIV-1 gag gene linked to nucleotides encoding human B cell and T cell epitopes of gp41, nef, gp120, RT, Tat and Rev into the cDNA clone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ) G22E/M48R+M51R. It also illustrates the cloning of HIV-1 pol and env genes into the cDNA clone of rVSV_(Ind) G21E/L111F/M51R and rVSV_(NJ) G22E/M48R+M51R.

FIG. 24 illustrates a Western blot analysis of BHK21 cells infected with rVSV_(Ind) G21E/L111F/M51R expressing Gag A, B, C, En, and RT and incubated at 31° C. and 37° C.

FIG. 25 illustrates a Western blot analysis of BHK21 cells infected with rVSV_(NJ) G22E/M48R+M51R expressing Gag A, B, C, En, and RT and incubated at 31° C. and 37° C.

FIG. 26 illustrates a Western blot analysis of BHK21 cells infected with rVSV_(Ind) G21E/L111F/M51R or rVSV_(NJ) G22E/M48R+M51R expressing HIV-1 pol gene.

FIG. 27 illustrates a Western blot analysis of BHK21 cells infected with rVSV_(Ind) G21E/L111F/M51R or rVSV_(NJ) G22E/M48R+M51R expressing HIV-1 gp160mss gene.

FIGS. 28A to 28F are graphs illustrating VSV N and HIV-1 protein peptide specific CD8+ T cell responses among the different vaccination groups of Table 6: G1 (FIG. 28A), G2 (FIG. 28B), G3 (FIG. 28C), G4 (FIG. 28D), G5 (FIG. 28E) and G5 without Env P18 (FIG. 28F).

FIG. 29 is a graph illustrating VSV N and HIV-1 protein peptide specific CD8+ T cell responses in vaccination group G6 of Table 6. The amino acid sequences of VSV N peptide and peptides from HIV-1 proteins that are used to stimulated the splenocytes are shown (SEQ ID NOS: 32-37).

FIGS. 30A and 30B illustrate the humoral immune responses against HIV-1 Gag (panel A) and Gp120 (panel B) induced in vaccination groups of Table 6, which were determined by Enzyme Linked Immunosorbant Assay (ELISA).

FIG. 31 depicts the HCV structural protein genes cloned into rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) and rVSV_(NJ)(GLM).

FIGS. 32A to 32D illustrate Western blot analyses of the expression of HCV Core (panel A), E1 (panel B), E2 (panel C), and NS4B (panel D) proteins from the rVSV_(Ind)(GLM), rVSV_(NJ)(GM), rVSV_(NJ)(GLM).

FIG. 33 depicts the HCV non-structural protein genes cloned into rVSV_(Ind)(GLM), rVSV_(NJ)(GM), and rVSV_(NJ)(GLM).

FIGS. 34A to 34C are Western blot analyses of the expression of HCV NS3 at 37° C. and 31° C. (FIG. 34A), NS5A at 37° C. (FIG. 34B), and NS5B at 37° C. and 31° C. (FIG. 34C) from the rVSV_(Ind)(GLM).

FIGS. 35A and 35B demonstrate the expression of HCV NS3, NS4B, NS5A, and NS5B from rVSV_(NJ)(GM) at 37° C. (FIG. 35A) and the expression of HCV NS5AB at 37° C. (FIG. 35B). The protein expression was detected by Western blot analysis.

DETAILED DESCRIPTION OF THE INVENTION

Overview

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, unless indicated otherwise, except within the claims, the use of “or” includes “and” and vice versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (for example “including”, “having” and “comprising” typically indicate “including without limitation”). Singular forms including in the claims such as “a”, “an” and “the” include the plural reference unless expressly stated otherwise. “Consisting essentially of” means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. “Consisting of” means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

All numerical designations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied (+) or (−) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”.

The term “administering” includes any method of delivery of a compound of the present invention, including a pharmaceutical composition, vaccine or therapeutic agent, into a subject's system or to a particular region in or on a subject. The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. “Parenteral administration” and “administered parenterally” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The term “amino acid” is known in the art. In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For the amino acids relevant to the present invention the designations are: M: methionine, R: arginine, G: glycine, E: glutamic acid, L: leucine, F: phenylalanine. In certain embodiments, the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

The term “antibody” as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), including polyclonal, monoclonal, recombinant and humanized antibodies and fragments thereof which specifically recognize and are able to bind an epitope of a protein. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Nonlimiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFvs may be covalently or non-covalently linked to form antibodies having two or more binding sites.

As used herein, the term “epitopes” refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in an animal, preferably in a mammal. An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that elicits an immune response in an animal. An epitope having antigenic activity is a site or fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays.

As used herein, the term “fragment” in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment, a fragment of a full-length protein retains activity of the full-length protein, e.g., immunogenic activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., non-immunogenic activity.

As used herein, the term “fragment” in the context of a nucleic acid refers to a nucleic acid comprising an nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least contiguous 80 nucleotides, at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains activity of the full-length protein, e.g., immunogenic activity. In another embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein, e.g., non-immunogenic activity.

The term “essentially noncytolytic” as used herein means that the recombinant vesicular stomatitis virus (rVSV) does not significantly damage or kill the cells it infects.

The term “HIV” is known to one skilled in the art to refer to Human Immunodeficiency Virus. There are two types of HIV: HIV-1 and HIV-2. There are many different strains of HIV-1. The strains of HIV-1 can be classified into three groups: the “major” group M, the “outlier” group 0 and the “new” group N. These three groups may represent three separate introductions of simian immunodeficiency virus into humans. Within the M-group there are at least ten subtypes or clades: e.g., clade A, B, C, D, E, F, G, H, I, J, and K. A “clade” is a group of organisms, such as a species, whose members share homologous features derived from a common ancestor. Any reference to HIV-1 in this application includes all of these strains.

The term “non-infectious” means of reduced to non-existent ability to infect.

As used herein, the terms “subject” or “patient” are used interchangeably. As used herein, the terms “subject” and “subjects” refers to either a human or non-human animal.

The term “pharmaceutical delivery device” refers to any device that may be used to administer a therapeutic agent or agents to a subject. Non-limiting examples of pharmaceutical delivery devices include hypodermic syringes, multichamber syringes, stents, catheters, transcutaneous patches, microneedles, microabraders, and implantable controlled release devices. In one embodiment, the term “pharmaceutical delivery device” refers to a dual-chambered syringe capable of mixing two compounds prior to injection.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin, which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement. An “oligonucleotide” refers to a single stranded polynucleotide having less than about 100 nucleotides, less than about, e.g., 75, 50, 25, or 10 nucleotides.

The terms “polypeptide”, “peptide” and “protein” (if single chain) are used interchangeably herein to refer to polymers of amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

In certain embodiments, polypeptides of the invention may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Chemical synthesis of polypeptides of the invention may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules (see e.g., U.S. Pat. Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193; K. Rajarathnam, et al., Science (1994): vol. 264, p 90; R. E. Offord, “Chemical Approaches to Protein Engineering”, in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852; L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994) 91: 12544-12548; M. Schnlzer, et al., Science (1992): vol., 3256, p 221; and K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985) 33: 184).

“VSV” is used to refer to vesicular stomatitis virus.

“rVSV” is used to refer to a recombinant vesicular stomatitis virus.

The term “Indiana”, and “IND” are used to refer to the VSV serotype Indiana (VSV_(Ind)).

The term “New Jersey”, and “NJ” are used to refer to the VSV serotype New Jersey (VSV_(NJ)).

“MWT” “M(WT)” are used to refer to a wild type M protein. The nucleotide sequence of wild type M gene of the VSV_(Ind) may comprise the nucleotide sequence represented by SEQ ID NO: 1. The amino acid sequence of wild type M protein of the VSV_(Ind) may comprise the amino acid sequence represented by SEQ ID NO: 3. The nucleotide sequence of wild type M gene of the VSV_(NJ) may comprise the nucleotide sequence represented by SEQ ID NO: 5. The amino acid sequence of wild type M protein of the VSV_(NJ) may comprise the amino acid sequence represented by SEQ ID NO: 8. “M51R” is used to refer to an M(WT) in the VSV_(Ind) having a methionine changed to an arginine at position 51. “G21E” is used to refer to an M(WT) in VSV_(Ind) having a glycine changed to a glutamic acid at position 21. “L111F” is used to refer to an M(WT) in VSV_(Ind) having a leucine changed to a phenylalanine at position 111. “G22E” is used to refer to an M(WT) in VSV_(NJ) having a glycine (G) changed to glutamic acid (E) at position 22. “L110F” is used to refer to an M(WT) in VSV_(NJ) having a leucine (L) changed to a phenylalanine (F) at position 110. “M48R+M51R” is used to refer to an M(WT) in VSV_(NJ) having a methionine (M) changed to an arginine (R) at positions 48 and 51. “rVSV_(Ind) M(G21E/L111F/M51R)” or “rVSV_(Ind) (GLM)” are used to refer to a rVSV_(Ind) having an M(WT) having a glycine changed to a glutamic acid at position 21, a leucine changed to a phenylalanine at position 111 and a methionine changed to an arginine at position 51. “rVSV_(NJ) M(G22E/M48R+M51R)” or “rVSV_(NJ) (GM)” are used to refer to rVSV_(NJ) having an M(WT) having a glycine changed to a glutamic acid at position 22 and a methionine changed to an arginine at positions 48 and 51, and “rVSV_(NJ) M(G22E/L110F/M48R+M51R)” or “rVSV_(NJ) (GLM)” are used to refer to rVSV_(NJ) having an M(WT) having a glycine changed to a glutamic acid at position 22, a leucine changed to a phenylalanine at position 110 and a methionine changed to an arginine at positions 48 and 51.

Overview

The inventors generated novel M proteins and novel attenuated rVSVs capable of producing the novel M proteins. The novel proteins of the present invention may include: M(G21E/L111F/M51R), M(G22E/M48R+M51R), and M(G22E/L110F/M48R+M51R). The novel attenuated rVSVs of the present invention may be used as protein expression and vaccine vectors and in methods for preventing or treating infections. The rVSV of the present invention may be applied to make vaccines for the infectious diseases of human and other animals to induce cellular and humoral immune responses.

Isolated Proteins and M Proteins

In one embodiment, the present invention relates to isolated proteins and to nucleotide sequences that encode the isolated proteins. As such, in one embodiment the present invention relates to an isolated peptide comprising an amino acid sequence selected from amino acid sequences listed as SEQ ID NOs: 4, 9 and 10. In another embodiment, the present invention relates to isolated nucleotide sequences comprising a nucleotide sequence selected from the polynucleotides listed as SEQ ID NOs: 2, 6 and 7.

In one embodiment, the present invention relates to novel VSV M proteins having at least one of the following substitutions: M(G21E/L111F/M51R), M(G22E/M48R+M51R), and M(G22E/L110F/M48R+M51R). In one aspect the present invention relates to a VSV M protein comprising an amino acid sequence selected from the amino acid sequences listed as SEQ ID NOs: 4, 9 and 10. In another embodiment the present invention relates to nucleotide sequences which encode for the novel VSV M proteins of the present invention. The nucleotide sequences may be selected from the group of sequences listed as SEQ ID NOs: 2, 6 and 7.

Methods of Preventing or Treating an Infection

Provided are methods of inducing an immune response, preventing or treating infections. In one embodiment, the methods may include administering to a subject: (a) an effective amount of a vaccine comprising an attenuated rVSV of one serotype having (i) a first modified M protein, the first modified M protein comprising the amino acid sequence of SEQ ID NO: 3 including at least the following substitutions: G21E/L111F/M51R, and (ii) an epitope of the pathogen; and (b) an effective amount of another vaccine comprising an attenuated rVSV of another serotype having: (i) a second modified M protein, the second modified M protein comprising the amino acid sequence of SEQ ID NO: 8 including at least the following substitutions: G22E/M48R+M51R, and (ii) the epitope of the pathogen. In embodiments of the present invention, the methods may include administering to a subject (a) an effective amount of rVSV_(Ind) M(G21E/L111F/M51R), and (b) an effective amount of either a rVSV_(NJ) M(G22E/M48R+M51R) or rVSV_(NJ) M(G22E/L110F/M48R+M51R) in a prime-boost immunization modality.

The term “effective amount” as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired result.

In certain embodiments, (a) is administered to the subject before (b) is administered to the subject.

In certain embodiments, (b) is administered to the subject more than one time over the course of treating or preventing.

In certain embodiments, (a) is administered to the subject in need thereof and (b) is administered to the subject in need thereof at about weeks three, eight and sixteen post-administration of (a).

In certain embodiments, (b) is administered to the subject before (a) is administered to the subject.

In certain embodiments, (a) is administered to the subject more than one time over the course of treating or preventing.

In certain embodiments, (b) is administered to the subject in need thereof and (a) is administered to the subject in need thereof at about weeks three, eight and sixteen post-administration of (b).

Recombinant Virus

In certain embodiments, present invention relates to a recombinant vesicular stomatitis virus (rVSV) which may be a full length VSV, essentially non-cytolytic, avirulent, capable of inducing an immune response in a subject, capable of reproducing virus particles to a high tire at permissive temperatures, reproducing virus particles to a low titre at semi-permissive temperatures and which may be incapable of producing virus at non-permissive temperatures, and that can express an epitope of a foreign pathogen. The rVSV of the present invention may be capable of inducing humoral, cellular and mucosal immune responses.

In one embodiment, the present invention relates to rVSV_(Ind) and rVSV_(NJ). The rVSV_(Ind) may be a full length, essentially noncytolytic rVSV_(Ind) M(G21E/L111F/M51R) capable of producing virus particles at a permissible temperature of about 31° C., and which may be incapable of or poorly capable of producing virus particles at a semi-permissive temperatures of about 37° C. and incapable of producing virus particles at non-permissive temperatures above 37° C., for example 39° C. In certain embodiments, the rVSV_(Ind) may include a M(G21E/L111F/M51R). In certain embodiments, the rVSV_(Ind) may include an M gene comprising a nucleotide sequence SEQ ID NO: 2.

In certain embodiments, the rVSV is a full-length, essentially noncytolytic rVSV_(NJ) M (G22E/M48R+M51R) or M(G22/L110F/M48R+M51R). In certain embodiments, the rVSV is an essentially noncytolytic rVSV_(NJ) including an M gene, wherein the nucleotide sequence of the M gene is selected from SEQ ID NO: 6 and SEQ ID NO: 7.

The rVSVs of the present invention can be prepared using techniques known in the art. In one embodiment, the rVSVs may be introduced in a host cell under conditions suitable for the replication and expression of the rVSV in the host. Accordingly, the present invention also provides a cell having a rVSV_(Ind) wherein the amino acid sequence of the virus' M protein is modified to provide an essentially non-cytotoxic which also allows the rVSV_(Ind) to effectively replicate at permissible temperature but may not replicate at non-permissible temperature.

As such, the present invention relates also to a cell having one or more of the recombinant VSVs of the present invention.

Vaccines or Immunogenic Compositions of the Invention

The present invention further features vaccines or immunogenic compositions comprising one or more of the rVSVs of the present invention. In one embodiment, the present invention features vaccines or immunogenic compositions comprising an rVSV_(Ind) and vaccines or immunogenic compositions comprising an rVSV_(NJ), as described above.

In one embodiment, the vaccines may include rVSVs expressing an epitope of a pathogen. In another embodiment, the vaccines may include a mixture or cocktail of rVSVs expressing different epitopes of a pathogen (see Table 6, vaccination groups 5 and 6).

The vaccine or immunogenic compositions of the invention are suitable for administration to subjects in a biologically compatible form in vivo. The expression “biologically compatible form suitable for administration in vivo” as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances maybe administered to any animal or subject, preferably humans. The vaccines of the present invention may be provided as a lyophilized preparation. The vaccines of the present invention may also be provided as a solution that can be frozen for transportation. Additionally, the vaccines may contain suitable preservatives such as glycerol or may be formulated without preservatives. If appropriate (i.e. no damage to the VSV in the vaccine), the vaccines may also contain suitable diluents, adjuvants and/or carriers.

The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.

Kits

The present invention provides kits, for example for preventing or treating an infection. For example, a kit may comprise one or more pharmaceutical compositions or vaccines as described above and optionally instructions for their use. In still other embodiments, the invention provides kits comprising one or more pharmaceutical compositions or vaccines and one or more devices for accomplishing administration of such compositions.

Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.

Advantages and Unique Features of the rVSVs of the Present Invention

Novel and Unusual Features of the Invention:

Normal assembly and release of rVSV_(Ind) M(G21E/L111F/M51) at permissive temperature (about 31° C.) made it possible to amplify the new mutant rVSVs at the permissible temperature to a high titre to make viral stock. The assembly defectiveness of rVSV_(Ind) M(G21E/L111F/M51) at non-permissive temperature (about 37° C. (around body temperature) increased the safety of the using the rVSV in human and other animals by significantly reducing the number of progeny infectious viruses at the non-permissive temperature. The addition of these mutations to the pre-existing M51R mutation in the M protein of rVSV_(Ind) further attenuated the virulence of VSV_(Ind).

The three mutations, M(G22E/M48R+M51R) or four mutations, M(G22E/L110F/M48R+M51R) in the M protein of rVSV_(NJ) did not make the virus temperature sensitive for the assembly of the virus. However, the addition of G22E mutation or G22E/L110F to the M48R+51R mutations made the VSV_(NJ) more attenuated and became significantly less pathogenic.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES

The present invention is further illustrated by the following examples which should not be construed as limiting in any way.

Example 1—Introduction of Mutations into the M Genes of rVSV_(Ind) and rVSV_(NJ) and Recovery of Recombinant VSV by Reverse Genetics

Mutations were introduced into the M gene of VSV_(Ind) (FIG. 1) and VSV_(NJ) (FIG. 2). Nucleotide sequences encoding the amino acids at each position were mutated by the mega-primer PCR method. Each mutation is expressed as a substitution of an amino acid at a specific position (e.g., M51 in M51R) with another amino acid (e.g., R in M51R). In order to attenuate further the virulence of VSV, the inventors combined mutations (G21E and L11F) in the tsO23 with methionine to arginine mutations (M51R) in the M gene, which reduced inhibitory activity of M protein on the cellular protein synthesis, in addition, reduced the assembly of the VSV particles at non-permissive (39° C.) and semi-permissive (37° C.) temperatures. The nucleotide sequences and amino acid changes from wild type to mutants in the M genes of rVSV_(Ind) and rVSV_(NJ) are shown in Tables 2, 3, 4 and 5. The changed nucleotide codons are underlined and changed nucleotide sequences and amino acid sequences are bold-faced.

Wild type and mutant recombinant vesicular stomatitis viruses (rVSV) were recovered from the cDNA plasmids by reverse genetics (FIG. 3). The VSV reverse genetics employs the BHK21 cells expressing DNA dependant RNA polymerase of bacteriophage T7 (T7) and a plasmid which encodes full length genomic RNA of VSV (pVSV) and 3 plasmids expressing nucleocapsid protein (pN), phosphoprotein (pP), and VSV polymerase L protein (pL). The transcription of the full length genomic RNA and the messenger RNAs for N, P, and L proteins are under the control of T7 RNA polymerase. Internal ribosome entry site (IRES) at the upstream of each VSV N, P, and L gene enhances the translation of proteins. The plasmids are transfected into BHK-T7 cells with Lipofectamine™ 2000 in concentrations of 10 μg of pN, 10 μg of pP, μ5 g of pL, and 15 μg of pVSV. The culture media from the transfected cells were harvested when the cells showed about 50-70% of CPE.

Example 2—the Mutation, L111F in the M Gene Significantly Reduces the Burst Sizes of rVSV_(Ind) at Semi-Permissive Temperature, 37° C.

The recovered viruses were purified 3 times by plaque picking and were amplified for a larger volume of stock viruses by infecting BHK21 cells with an MOI of 0.1 at 31° C. The inventors infected BHK21 cells and human neuroblastoma cells, SH-SY5Y with an MOI of 3 of rVSVs. The infected cells were incubated at permissive temperature (31° C.) and semi-permissive temperature (37° C., body temperature) to determine the temperature sensitivity of the new M mutants in the assembly of virus particles. Culture media from the infected cells were collected every 2 hours until 10 hours after the infection, and the number of infectious viral particles in the culture media was determined by plaque assay with Vero E6 cells. The cells infected with the mutant viruses for the plaque assay were incubated at 31° C. Wild type and all mutants replicated equally well and produced similar titre of infectious viruses all along the period of 10 hrs of infection (FIG. 4A and FIG. 4C). However, the mutants of rVSV_(Ind), rVSV_(Ind)-G21E/L111F and rVSV_(Ind)-G21E/L111F/M51R replicated in significantly lower titre than wild type or M51R mutant of rVSV_(Ind) at 37° C. The differences in producing infectious particles between the wild type and the new M mutant, rVSV_(Ind)-G21E/L111F/M51R were as large as four logs (FIG. 4B and FIG. 4D). The error bars in FIG. 4 represent standard error of the mean. The P values for viral titers of rVSV_(Ind)-G21E/L111F/M51R at 4 hrs, 6 hrs, 8 hrs, and 10 hrs postinfection obtained by comparing them to titers of rVSV_(Ind)-WT are p<0.0001, p=0.0055, p=0.0040, and p=0.0015 in FIG. 4B. The P values for viral titers of rVSV_(Ind)-G21E/L111F at 4 hrs, 6 hrs, 8 hrs, and 10 hrs postinfection obtained by comparing them to titers of rVSV_(Ind)-WT are p<0.0001, p=0.0055, v0.0041, and p=0.0016 in FIG. 4B. The P values in FIG. 4D were <0.005. P values were computed by using two-sided independent t tests.

Example 3—Mutations in the M Gene of rVSV_(NJ), G22E and L110F do not Reduce the Burst Size of the rVSV_(NJ) at 37° C.

BHK21 cells were infected with an MOI of 3 of rVSV_(NJ), wild type and M gene mutants, incubated at both 37° C. (semi-permissive temperature) and 31° C. (permissive temperature) and the culture media was harvested at 10 hrs post infection. The viral titer of each virus in the culture media was determined by plaque assay using Vero E6 cells. The average viral titre from the duplicate samples is shown in the table. Wild type and all M mutants of rVSV_(NJ) replicated equally well at both temperatures 31° C. and 37° C. (FIG. 5), indicating the introduction of G22E and L110F mutations into the M gene of rVSV_(NJ) did not affect the assembly and release of the virus at 37° C. The error bars in FIG. 5 represent standard error of the mean.

Example 4—Assembly Defectiveness at Semi-Permissive Temperature by L111F Mutation in the M Gene of rVSV_(Ind) does not Affect the Expression of VSV Proteins

BHK21 cells were infected with an MOI of 6 of rVSV_(Ind) and rVSV_(NJ), wild type and M gene mutants. The infected cells were incubated at both 37° C. and 31° C. for 6 hrs. The infected cells were lysed at 6 hrs post-infection, 5 μg of total protein was loaded to the SDS-PAGE gel, and rVSV proteins were detected by Western blot analysis using rabbit antiserum against VSV_(Ind) and VSV_(NJ) (1:5000 dilution). The result demonstrate that in spite of the mutations that reduced the burst size of the rVSV_(Ind) at 37° C. (L111F in G21E/L111F and G21E/L111F/M51R), the level of VSV protein expression is comparable to the wild type rVSV_(Ind) (FIG. 6A). Wild type and all M mutants of rVSV_(NJ) expressed their proteins in a similar level at both 31° C. and 37° C. (FIG. 6B).

Example 5—Combined Mutations, G21E/L111F/M51R in the M Gene of rVSV_(Ind) Reduced Cytopathogenicity Significantly in BHK21 Cells and Human Neuroblastoma Cells, SH-SY5Y

In order to examine the effects of the mutations, L111F and M51R of M gene of rVSV_(Ind) on the cytopathogenicity, the inventors infected BHK21 cells (FIG. 7) and human neuroblastoma cells (FIG. 8) with an MOI of 0.1 of rVSV_(Ind). The typical cytopathic effects by the VSV are rounding-up of infected cells and cell lysis. At 20 hrs after infection, the cytopathic effect caused by the rVSV_(Ind)-G21E/L111F/M51R was compared with those by the other rVSV_(Ind). In 20 hrs of infection with the rVSV_(Ind)-G21E/L111F/M51R mutant showed the most reduced cytopathic effects, none or small number of round-up cells at 37° C. and the combination of the mutations L111F and M51R (FIG. 7N and FIG. 8L) further attenuated the cytopathogenicity of the virus than the single mutation of each in both BHK21 and SH-SY5Y cells.

Example 6—Reduced Cytopathic Effects in the BHK21 Cell by the rVSV_(NJ) with the M Gene Mutations, G22E/M48R+M51R and G22E/L110F/M48R+M51R

In order to examine the effects of the mutations, G22E, L110F and M48R+M51R of M gene of rVSV_(NJ) on the cytopathogenicity, the inventors infected BHK21 cells and human neuroblastoma cells with an MOI of 0.1 of rVSV_(NJ). At 20 hrs after infection, the inventors compared the cytopathic effects (cell round-up and lysis) caused by the rVSV_(NJ) wild type and other M mutants. In 20 hrs of infection with the G22E/M48R+M51R mutant and G22E/L110F/M48R+M51R mutant showed the least cytopathic effects at 37° C. and the combination of the mutations, G22E and M48R+M51R further attenuated the cytopathogenicity of the rVSV_(NJ) in both BHK21 (FIGS. 9L and 9N) and SH-SY5Y cells (FIGS. 10L and 10N).

Example 7—rVSV_(Ind) with the Mutations of G21E/L111F/M51R and rVSV_(NJ) with the Mutations of G22E/M48R+M51R and G22E/L110F/1V148R+M51R in the M Protein was Attenuated to a Degree that Mice Injected with the Virus into the Brain Showed No Neurological Signs or any Symptoms Such as Weight Loss

VSV does not show the neurotropism in host animal during the natural infection through the skin abrasion or sandfly or mosquito bites. Nevertheless, when wild type VSV is injected directly into the nose or brain in mice or monkeys, the animals demonstrate neurological symptoms. In order to examine the neurovirulence of the new M mutants of VSVs, the inventors injected Swiss Webster mice with 1×10⁶ PFU of mutant VSV and 1×10³ PFU of wild type VSV into the intralateral ventricle of the brain. The inventors purchased 5-week-old Swiss Webster mice with intralateral ventricular implant from Charles River laboratory. Three mice/group were injected with viruses after one week of arrival to the animal facility. After the viral injection, mice were observed for neurological signs and were weighed every two days for 4 weeks. The mice injected with 1×10³ PFU of wild type rVSV_(Ind) died within four days after injection (FIG. 11B). Two mice injected with 1×10⁶ PFU of rVSV_(Ind)-M51R (FIG. 11C) lost about 20% of body weights 6 days after injection and the body weight bounced back to normal weight at about 14 days after injection. One mice injected with the rVSV_(Ind)-M51R did not lose the body weight through the experiment. All three mice injected with rVSV_(Ind)-G21E/L111F/M51R showed no sign of illness and did not lose their body weight for 4 weeks until the mice were sacrificed (FIG. 11D) indicating the combination of mutations, G21E/L111F and M51R significantly attenuated the rVSV_(Ind) and lost its neurovirulence in mice. One mice injected with rVSV_(NJ)-WT showed paralysis in both hind legs and it was sacrificed on day 6 after injection (FIG. 12A). Two other mice injected with rVSV_(NJ)-WT and mice injected with rVSV_(NJ)-M48R+M51R, rVSV_(NJ)-G22E/M48R+M51R, or rVSV_(NJ)-G22E/L110F/M48R+M51R showed no sign of illness and weight loss during 4 weeks after injection (FIGS. 12B, 12C, and 12D). The error bars in FIGS. 11 and 12 represent standard error of the mean.

Example 8—Generation of rVSVs Expressing HIV-1 Gag Protein as a Gene of Interest; HIV-1 Gag Proteins are Expressed Equally Well at Both 31° C. and 37° C.

The newly generated M mutants, G21E/L111F/M51R mutant of rVSV_(Ind) and G22E/M48R+M51R and G22E/L110F/M48R+M51R mutants of rVSV_(NJ) demonstrated the reduced cytopathogenicity and comparable protein expressions to the wild type rVSV at 37° C. In order to be used as a vaccine vector, the new M mutants of rVSV should induce good immune responses in vivo, both humoral and cellular immune responses. When it is expressed alone in cells, HIV-1 gag proteins produce virus like particles and the virus like particles are secreted from the cells. Therefore, the gag protein was suitable protein to express from the new M mutants of rVSV to examine both cellular and humoral immune response. In addition, the CD8+ cytotoxic T cell epitope, H-2K^(d)-restricted peptide (NH₂-AMQMLKETI-COOH) (SEQ ID NO: 33) in the HIV-1 Gag is well studied in the Balb/c mouse. The inventors inserted the full-length HIV-1 gag gene linked to conserved human CD8+ T cell epitopes from gp41, gp120, and nef protein of HIV-1 (Gag-En). The Gag-En gene was inserted into the junction of G gene and L gene in the full-length cDNA clones of wild type (WT) and G21E/L111F/M51R (GLM) of rVSV_(Ind) and wild type, G22E/M48R+M51R (GM), and G22E/L110F/M48R+M51R(GLM) of rVSV_(NJ). The rVSVs were recovered from the cDNA clones by reverse genetics as described in FIG. 13A and the expression of Gag-En from the rVSVs was examined by Western blot analysis using monoclonal antibody against HIV-1 p24. For the Western blot analysis, BHK21 cells were infected with MOI of 6 of rVSVs and incubated at 31° C. and 37° C., and cell lysates were prepared at 6 hrs post-infection. Total protein of 5 μg was loaded onto the SDS-PAGE for the Western blot analysis. rVSV_(Ind)(GLM)-Gag-En, rVSV_(NJ)(GM)-Gag-En, and rVSV_(NJ)(GLM)-Gag-En expressed Gag-En protein (64 kDa) well at semi-permissive temperature (37° C., FIG. 13C) and the expression level was similar to that at permissive temperature (31° C., FIG. 13B).

Example 9—Vaccination Regimen with rVSV in Mice

Six Balb/c mice per group were vaccinated with the prime-boost regimen illustrated in FIG. 14. Mice were grouped according to the vaccine vector type (wild type vs. mutant) and regimen, e. g., priming and boosting with the same serotype of rVSV or alternating the two serotypes for priming and boost. The mice were prime-vaccinated intramuscularly with 5×10⁶ PFU of rVSVs at age of 6 weeks. Three weeks after the priming, mice were boost-vaccinated with the same dose of rVSV as the prime vaccination. A week after the booster injection, splenocytes and sera were collected to detect the HIV-1 Gag specific CD8+ T cell immune responses and humoral immune responses.

Example 10—Priming with rVSV_(Ind)(GLM)-HIV-1 Gag-En and Booster with rVSV_(NJ)(GLM)-HIV-1 Gag-En Induces Strongest CD8+ T Cell Immune Responses Against HIV-1 Gag

The T cells stimulated by the interaction with MHC I molecule on the antigen presenting cells loaded with the peptide enhances the secretion of interferon-γ (IFN-γ), which indicates the antigen specific T cell immune responses. The splenocytes were double stained with FITC-anti-CD8 and APC-anti-IFN-γ for CD8+ T cells with the increased intracellular INF-γ. In order to examine the HIV-1 Gag protein specific CD8+ T cell immune response, splenocytes were isolated and 1×10⁶ cells were stimulated with H-2K^(d)-restricted HIV-1 Gag peptide, NH₂-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molecules and stained with APC rat anti-mouse IFN-γ for intracellular IFN-γ. The secretion of IFN-γ was blocked with Brefeldin A before staining them. VSV specific CD8+ T cell immune responses were examined with the use of nucleocapsid specific peptide, IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide specific CD8+-IFN-γ+ T cells were counted with FACSCalibur, a flowcytometer. The splenocytes treated with DMSO (solvent for the peptide, FIG. 15) did not stimulate the CD8+ T cells indicating that treating the splenocytes with the VSV N peptides and HIV-1 Gag peptides stimulated specifically CD8+ T cells in FIG. 16 and FIG. 17. Prime and boost immunization by alternating two serotypes of rVSV(WT) or rVSV(GLM) mutants induced stronger CD8+ T cell immune responses against VSV as well as HIV-1 Gag proteins than using a single serotype of rVSV for the prime and boost vaccinations as seen in groups 1, 2, 5, 6, 9, and 10. (FIG. 16 and FIG. 17). With the vaccination with the new M mutants or rVSV, CD8+ T cell responses against HIV-1 Gag protein was induced better when mice were vaccinated with rVSV_(Ind)(GLM)-Gag for priming and rVSV_(NJ)(GLM)-Gag for boosting than vice versa (FIG. 17, group 6 vs. group 10). P value on the group 6 in FIG. 17 was computed by using two-sided independent sample t test, and the result was compared to that for the group 10. The error bars in FIGS. 16 and 17 represent standard error of the mean.

Example 11—HIV-1 Gag Specific Humoral Immune Responses

Generation of HIV-1 Gag specific antibody was examined with the serum collected at a week after the boost immunization. The Gag specific antibody titer was determined by the indirect enzyme-linked immunosorbent assay (ELISA). For the ELISA, 96 well ELISA plate was coated with recombinant p55 at a concentration of 125 ng/well. The mouse serum was diluted 1:100. The antibody bound to the antigen, p55 was detected with secondary antibody, sheep anti-mouse IgG-HRP. The enzymatic activity of HRP was detected by adding substrates, a mixture of hydrogen peroxide and tetramethylbenzidine. The OD of each sample was read at the wavelength of 450 with the microplate reader. The humoral immune responses against HIV-1 Gag (generation of antibody against HIV-1 Gag) was induced well in mice vaccinated with the new M mutants, and the best humoral immune responses against HIV-1 Gag were induced when two serotypes of rVSV(WT) or rVSV(GLM) were alternated for prime and booster injection (FIG. 18, groups 5, 6, 9, and 10). As for the utilization of the new M mutants for inducing humoral immune responses against foreign proteins, priming with rVSV_(Ind)(GLM)-Gag and booster with rVSV_(NJ)(GLM)-Gag worked better than vice versa (FIG. 18, group 6 vs. group 10). The error bars in FIG. 18 represent standard error of the mean.

Example 12—Increasing Doses of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), and rVSV_(NJ)(GLM) for Vaccination Induced Stronger Immune Responses in Mice

As illustrated in FIG. 17 and FIG. 18, stronger HIV-1 Gag specific CD8+ T cell immune responses and humoral immune responses were induced when mice were prime vaccinated with rVSV_(Ind)(GLM)-Gag and booster vaccinated with rVSV_(NJ)(GLM)-Gag. However the immune responses were not as strong as those induced with rVSV_(Ind)(WT)-Gag and rVSV_(NJ)(WT)-Gag. Therefore, the inventors wanted to examine whether they could increase the immune responses, if they increase the doses of the rVSV_(Ind)(GLM)-Gag and rVSV_(NJ)(GLM)-Gag. Six (6) week old Balb/c mice, 6 mice/group, were vaccinated with rVSV_(Ind)(GLM)-Gag for priming and rVSV_(NJ)(GLM)-Gag or rVSV_(NJ)(GM)-Gag for booster. Because the rVSV_(NJ)(GM) was as attenuated as the rVSV_(NJ)(GLM) in vitro and in vivo, the inventors included rVSV_(NJ)(GM)-Gag as a booster virus, and compared the immune responses to that induced by rVSV_(NJ)(GLM)-Gag. Mice groups were vaccinated with various doses of rVSV_(Ind)(GLM)-Gag, rVSV_(NJ)(GLM)-Gag, and rVSV_(NJ)(GLM)-Gag; 5×10⁶ PFU/mouse, 5×10⁷ PFU/mouse, 5×10⁸ PFU/mouse, and 5×10⁹ PFU/mouse (Table 1).

Mice were vaccinated according to the schedule as seen in FIG. 14 and were sacrificed a week after booster vaccination for splenocytes and serum. The splenocytes were double stained with FITC-anti-CD8 and APC-anti-IFN-γ for CD8+ T cells with the increased intracellular INF-γ. In order to examine the HIV-1 Gag protein specific CD8+ T cell immune response, splenocytes were isolated and 1×10⁶ cells were stimulated with H-2Kd-restricted HIV-1 Gag peptide, NH2-AMQMLKETI-COOH (SEQ ID NO: 33), the cells were stained with FITC rat anti-mouse CD8 for the CD8 molecules and stained with APC rat anti-mouse IFN-γ for intracellular IFN-γ. The secretion of IFN-γ was blocked with Brefeldin A before staining them. VSV specific CD8+ T cell immune responses were examined with the use of nucleocapsid specific peptide, IN275: NH2-MPYLIDFGL-COOH (SEQ ID NO: 32). Peptide specific CD8+-IFN-γ+ T cells were counted with FACSCalibur, a flowcytometer.

CD8+ T cell immune responses against VSV N protein were very similar in all vaccination groups with different doses of rVSV_(Ind)(GLM)-Gag, rVSV_(NJ)(GLM)-Gag, or rVSV_(NJ)(GM)-Gag (FIG. 19). The HIV-1 Gag specific CD8+ T cell immune responses after prime vaccination with rVSV_(Ind)(GLM)-Gag and booster with rVSV_(NJ)(GLM)-Gag looked increased a little bit when the vaccine doses were increased, but differences were not statistically significant (FIG. 20, groups 2, 3, 4, 5). However, when mice were prime vaccinated with rVSV_(Ind)(GLM)-Gag and booster vaccinated with rVSV_(NJ)(GM)-Gag, the frequency of the HIV-1 Gag specific CD8+ T cells were increased significantly with the increasing dose of 5×10⁸ PFU. The HIV-1 Gag specific CD8+ T cell response was strongest in a group vaccinated with 5×10⁹ PFU/mouse (FIG. 20. groups 6, 7, 8, 9). P values on the groups 8 and 9 in FIG. 20 were computed by using two-sided independent sample t test, the result of group 8 was compared to that for the group 7, and the result of group 9 was compared to that for the group 8. The error bars in FIGS. 19 and 20 represent standard error of the mean.

Generation of antibody against HIV-1 Gag protein was increased with the increasing doses of rVSV_(Ind)(GLM)-Gag for priming and booster with rVSV_(NJ)(GLM)-Gag or rVSV_(NJ)(GM)-Gag (FIG. 21). Booster vaccination with rVSV_(NJ)(GLM)-Gag or with rVSV_(NJ)(GM)-Gag generated similar level of Gag specific antibodies in various doses of the virus. The error bars in FIG. 21 represents standard error of the mean.

Example 13—Generation of rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) Expressing HIV-1 Proteins; Gag, Pol, Env, and RT

For HIV-1 vaccines utilizing the rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) as vaccine vectors, the inventors included HIV genes encoding most of the large polyproteins, which are cleaved into functional proteins-Gag and Env and proteins with enzymatic activities (pol gene products). In addition, HIV-1 gag gene was linked to nucleotides encoding peptide epitopes for T cells and B cells in humans. The inventors have generated rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) carrying cassettes encoding HIV-1 Gag linked to B-cell epitopes from HIV Env protein derived from multiple viral clades (FIG. 22). The inventors have generated the rVSV_(Ind)(GLM)-Gag-En and rVSV_(NJ)(GM)-Gag-En, which expresses gag gene and T cell peptide epitopes from Nef, Gp120, and Gp41 (FIG. 23). The inventors generated rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) with HIV-1 Gag-RT, which encodes gag protein with peptide epitopes from RT, Tat, and Rev proteins (FIG. 23). In addition, the inventors generated rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) with pol gene and env gene, respectively (FIG. 23). Previously, the inventors have demonstrated that replacement of signal peptide of HIV-1 envelope protein with the honeybee melittin signal peptide dramatically increased the rates of Gp120 expression, glycosylation, and secretion (Yan Li et al., PNAS, 93: 9606-9611, 1996). The increased expression and secretion of Gp120 are expected to enhance the induction of the T cell and B cell immune responses against HIV-1 gp120. Therefore, the inventors generated rVSVs with HIV-1 env gene with melittin signal sequence (gp160mss). The recombinant VSVs with the HIV-1 proteins were plaque purified and were amplified for the stock viruses. The expression of HIV-1 proteins at 31° C. and 37° C. from the mutant VSV was examined by Western blot analysis. The Gag proteins tagged with T cell and B cell epitopes from various HIV-1 proteins were expressed well from rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) at both 31° C. and 37° C. (FIG. 24 and FIG. 25). The cloned HIV-1 pol gene in the rVSV genome encodes a polyprotein, which is cleaved into protease (PR, p11), reverse transcriptase (RT, p66/p51), and integrase (IN, p31). The inventors detected two subunits of RT products, p66 and p51. The p51 is generated from the p66 by proteolytic cleavage of a fragment of p15 at the carboxy terminus. The RT products p66 and p51 were expressed equally well from both rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) and at both 31° C. and 37° C. (FIG. 26). The env gene encodes glycoprotein Gp160, which is cleaved into Gp120 of receptor binding protein and gp41 of fusion inducing cytoplasmic and transmembrane protein. Gp160 is cleaved by the cellular trans-golgi resident protease furin. The Gp160 expressed in BHK21 cells from the rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) was not cleaved to Gp120 and Gp41 or the cleavage was not very efficient, although the level of expression was good at both 31° C. and 37° C. (FIG. 27). Our previous data on the expression of the Gp160 in BHK21 cells and cleavage into Gp120 and Gp41 demonstrated that the processing was not very efficient (Kunyu Wu et al., Journal of General Virol., 90:1135-1140, 2009).

Example 14—Immunization Studies in Mice with the rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) with HIV-1 Proteins, Gag, Gp160 and RT

The preliminary prime-boost immunization studies with new M mutant rVSVs, rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) revealed that priming mice with the rVSV_(Ind)(GLM) and boosting with rVSV_(NJ)(GM) induced better CD8+ T cell immune response (FIG. 17) and humoral immune responses (FIG. 18) against the foreign gene than the other way around. With the rVSV_(Ind)(GLM)-Gag priming and rVSV_(NJ)(GM)-Gag boosting, the dosage of 5×10⁸ pfu of each virus induced better Gag specific CD8+ T cell responses than lower dosages, although the B cell responses against Gag was similar to that induced with the lower dosages (FIG. 20 and FIG. 21). The inventors chose 5×10⁸ pfu as a dosage for immunization studies with rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) expressing HIV-1 Gag, Gp160, and RT.

The inventors prime immunized Balb/c mice with rVSV_(Ind)(GLM) expressing HIV-1 proteins and boost immunized with rVSV_(NJ)(GM) expressing HIV-1 proteins in three weeks after the priming as described in the table 6. The inventors examined how the immune responses against the individual HIV-1 proteins are induced with individual injection or with the mixed injection of three different viruses expressing separate proteins. One week after the boost immunization, the inventors sacrificed the immunized mice for their splenocytes and sera. The 1×10⁶ splenocytes were stimulated with H-2K^(d)-restricted HIV-1 protein specific peptides, which are Gag, Env P18, RT354, RT464, and RT472. The peptide sequences are shown in the FIG. 29. The stimulated splenocytes were double stained for the cell surface CD8 molecules and intracellular IFN-γ of T cells with FITC rat anti-mouse CD8 and APC rat anti-mouse IFN-γ. The regimen, prime-boost immunization with rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) induced peptide specific CD8+ T cell immune responses with different degree depending on the peptides the inventors used for the stimulation. CD8+ T cells against Env peptide stimulated CD8+ T cells the most up to 19%-23% of CD8+ T cells on average (FIG. 28C, G3 and FIG. 28E, G5). About 4.5% or 3% of CD8+ T cells were responsive to the Gag protein in mice immunized with rVSV expressing Gag alone (FIG. 28B, G2) or with mixed three rVSVs expressing Gag A, Gag B, and Gag C linked with various B and T cell epitopes of HIV-1 Gp120 and Gp41 (FIG. 29, G6). RT specific CD8+ T cells were generated in about 2% of CD8+ T cells in mice injected with rVSV with pol gene (FIG. 28D, G4). CD8+ T cell responses against individual HIV-1 protein, Gag and RT were stronger when mice were injected with single rVSV expressing individual protein (FIG. 28B, G2 and FIG. 28D, G4) than when injected with mixed rVSVs expressing single protein (FIG. 28E, G5). Env specific CD8+ T cell responses were strongest and were similar in both single injection and mixed injections. The CD8+ T cells responses against Env peptide epitope are immunodominant in Balb/c mice. Currently, the inventors are uncertain why mixed injections with three different rVSVs expressing Gag, Env, and RT induced comparatively lower CD8+ T cell responses against VSV N, gag, and RT than with the single virus injection. It is possible that the competition of each virus for the antigen presenting cells in the mixed virus injection lowered the CD8+ T cells against Gag and RT. Because rVSV is common vector for HIV-1 proteins in mice either injected with single rVSV or mixed rVSVs, VSV N supposed to induce similar or better CD8+ T cell responses in the mixed injection. However, the CD8+ T cell responses against VSV N in mice are lower even with the single injections when the Env is expressed (FIG. 28C, G3) as in the mixed injection (FIG. 28E, G5). Therefore, it may be the result of immune dominance of Env protein in Balb/c mice, which affects negatively to the presentation of other proteins to the CD8+ T cells when Env is expressed together. For the combined immunizations in mice, all three rVSVs were injected at the single site, which may have resulted in the competition of different proteins for the antigen presentation in the same antigen presentation cells. The inventors will test the immunization with the mixed injections at more than one injection sites to induce CD8+ T cell responses.

HIV-1 Gag proteins expressed in cells can form virus like particles (VLP) and the VLP can be released from the cells. The released VLPs can induce humoral immune responses against Gag proteins. HIV-1 env gene encodes glycosylated surface proteins, which are processed through ER-golgi network to be properly folded and cleaved into transmembrane subunit Gp41 and surface unit Gp120. The Gp41 and Gp120 are associated by non-covalent bond and mature to form a trimer of Gp41 and Gp120 heterodimer. The matured trimer of Gp41 and Gp120 are exported to the cell surface. The Gp120 tends to fall off from the cell surface because of the weak bondage between Gp41 and Gp120. Therefore, antibody against Gp120 can be induced to the cell surface Gp120 or fallen off Gp120. Antibody titer against HIV-1 Gag protein and Gp120 was determined by ELISA. For the Gag antibody the microplate was coated with 125 ng/well of recombinant p55 (Pierce Biotechnology, RP-4921) and 50 μl of mice sera was tested with the dilution of 1:100, 1:200, and 1:400. Gag antibody was produced in mice injected with rVSV Gag-En alone (FIG. 30A, G2) and with mixed viruses rVSV-Gag A+rVSV Gag B+rVSV Gag C (FIG. 30A, G6). For the Gp120 ELISA, 96 well microplate was coated with 250 ng/well of HIV-1 SF162 Gp140 trimer from clade B (NIH AIDS reagent program, cat#12026). Mice sera were diluted in 1:100, 1:200, and 1:400 and 50 μl of the diluted serum was added to the microplate for the ELISA. Antibody specific to Gp120 was generated in mice injected with rVSV-HIV-1 Gp160 alone and in mice injected with the mixed viruses expressing each protein of Gag, Gp160, and RT (FIG. 30B, G3 and G5). The titer of Gp120 antibody was slightly lower, although not statistically significant, in mice injected with mixed viruses than in mice injected with single virus expressing Gp160.

The newly generated M mutants of rVSVs, rVSV_(Ind)(GLM) and rVSV_(NJ)(GM) induced CD8+ T cell and humoral immune responses against various HIV-1 proteins expressed from the vector after injecting mice with an individual virus expressing single virus or with mixed viruses expressing various HIV-1 proteins.

Example 15—Generation of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), and rVSV_(NJ)(GLM) with Hepatitis C Virus Structural Proteins

Generally, the humoral immune response is the first line of defence mediated by adaptive immune responses against any pathogens. Although humoral immune responses against HCV and its role in the prevention of HCV infection is not well studied compared to the HCV specific cellular immune responses, it is worthy to include vaccines which can induce HCV specific antibodies. It has been demonstrated that nucleocapsid protein core and surface glycoproteins E1 and E2 form virus-like particles (VLP) which can be released from the cells (Blanchard, E. et al., J. Virol. 76:4073-4079, 2002). In addition, it has been demonstrated that HCV protein p7, a viroporin forming an ion channel in the ER membrane, takes part in releasing the HCV particles from the infected cell (Steinmann, E. et al., PLoS Pathogens 3:962-972, 2007). Another HCV transmembrane protein NS4B forms a membranous web structure, which mainly consists of the ER membrane. The membranous web formed by NS4B is a microstructure for the production of progeny HCV. It is not well known what functions NS4B has in the replication of HCV, but it is appealing to include NS4B into vaccine candidates simply because of its nature forming a membranous structure of ER in which other HCV proteins, especially Core, E1, E2, p7 are localized. Therefore, including Core, E1, E2, P7, and NS4B together into the vaccine may induce humoral and cellular immune responses.

For the HCV structural protein vaccines using the new M mutant rVSVs, the inventors inserted the HCV core gene, E1E2P7 and NS4B genes together (connected by the VSV intergenic junction sequences), and CoreE1E2P7 and NS4B genes together (connected by the VSV intergenic junction sequences) to the junction of VSV G gene and L (FIG. 31). The rVSVs; rVSV_(Ind)(GLM)-FC, rVSV_(Ind)(GLM)-CE1E2P7/NS4B, rVSV_(NJ)(GM)-FC, and rVSV_(NJ)(GLM)-E1E2P7/NS4B were generated by reverse genetics. The inventors had a trouble to generate the rVSV_(NJ)(GM) with CE1E2P7/NS4B from the plasmid DNA, therefore, the inventors removed the core and cloned E1E2P7/NS4B into the plasmid of rVSV_(NJ)(GLM) and generated the rVSV_(NJ)(GLM)-E1E2P7/NS4B. The expression of Core, E1, E2, and NS4B from the recombinant VSV at 31° C. and 37° C. was examined by Western blot analysis using antibodies against each protein (FIG. 32). The proteins were expressed similarly in quantity at both temperatures. Core (FIG. 32A), E1 (FIG. 32B), and E2 FIG. 32C) were expressed well from the rVSV_(Ind)(GLM)-CE1E2P7/NS4B, but the expression of NB4B was lower than the other proteins (FIG. 32D).

Example 16—Generation of rVSV_(Ind)(GLM), rVSV_(NJ)(GM), and rVSV_(NJ)(GLM) with HCV Non-Structural Proteins

The inventors want to target most of the HCV proteins including core, E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins in order to induce HCV specific CD8+ T cell and CD4+ T cell immune responses to multiple proteins. HCV nonstructural (NS) proteins-NS3, NS4A, NS4B, NS5A, and NS5B cover more than half of the HCV polyprotein. The NS proteins are cleaved into individual proteins by NS3 with the help of NS4A. Several studies demonstrate that patients who recover from the acute HCV infection develop strong CD4+ T cell and CD8+ T cell responses against multiple epitopes in the NS3 protein (Diepolder, H. M. J. Virol. 71:6011-6019, 1997; Lamonaca, V. et al. Hepatology 30:1088-1098, 1999; Shoukry, N. H. et al. J. Immunol. 172:483-492, 2004) indicating that including NS3 in the vaccine candidate is important to elicit successful cellular immune responses against HCV. NS3 protein, a serine protease and RNA helicase associates with NS4A and resides on the ER membrane (Sato, S. et al. J. Virol. 69:4255-4260, 1995; Failla, C. et al. J. Virol. 69:1769-1777, 1995). NS5A protein, a phosphoprotein is believed to be involved in HCV RNA synthesis together with NS5B protein, a RNA dependent RNA polymerase (Shirota, Y. et al., J. Biol. Chem., 277:11149-11155, 2002; Shimakami, et al. J. Virol. 78:2738-2748, 2004). NS5B protein is a RNA dependent RNA polymerase that synthesizes positive sense HCV genomic RNA as well as intermediate negative sense genomic RNA (Beherens, S. E. EMBO J. 15:12-22, 1996). NS5B protein is a tail-anchored protein and associates with ER membrane through its carboxyl terminal 20 amino acids (Yamashita, T. J. Biol. Chem. 273:15479-15486, 1998; Hagedorn, C. H. Curr. Top. Microbiol. Immunol. 242:225-260, 2000).

The inventors cloned HCV NS genes as a gene for a single protein or a gene for a polyprotein of 2 or 3 NS proteins. The NS genes NS3, NS34AB, NS5A, NS5B, NS5AB were cloned into the junction at G gene and L gene of pVSV_(Ind)(GLM) and pVSV_(NJ)(GM) (FIG. 33). The inventors generated rVSV_(Ind)(GLM)-NS3, rVSV_(Ind)(GLM)-NS34AB, rVSV_(Ind)(GLM)-NS5A, rVSV_(Ind)(GLM)-NS5B, and rVSV_(Ind)(GLM)-NS5AB, and the expression of the NS proteins at 31° C. and 37° C. were determined by Western blot analysis using antibodies against each NS protein (FIG. 34). The inventors generated rVSV_(NJ)(GM)-NS3, rVSV_(NJ)(GM)-NS4B, rVSV_(NJ)(GM)-NS34AB, rVSV_(NJ)(GM)-NS5A, rVSV_(NJ)(GM)-NS5B, and rVSV_(NJ)(GM)-NS5AB. The expression of each protein from the rVSV_(NJ) vector at 37° C. was examined by Western blot analysis using antibodies against each NS protein (FIG. 35). Although the expression level of NS proteins from the rVSV_(Ind) and rVSV_(NJ) was different, it was good enough to use them as HCV vaccine.

TABLE 1 Mouse Vaccination Groups and Regimen Prime Boost Vacci- Titer Titer nation (PFU)/ (PFU)/ # of Groups Viruses 50 μl Viruses 50 μl Mice 1 Ind (WT)-Gag 5 × 10⁶ NJ (WT)-Gag 5 × 10⁶ 6 2 Ind (GLM)-Gag 5 × 10⁶ NJ (GLM)-Gag 5 × 10⁶ 6 3 Ind (GLM)-Gag 5 × 10⁷ NJ (GLM)-Gag 5 × 10⁷ 6 4 Ind (GLM)-Gag 5 × 10⁸ NJ (GLM)-Gag 5 × 10⁸ 6 5 Ind (GLM)-Gag 5 × 10⁹ NJ (GLM)-Gag 5 × 10⁹ 6 6 Ind (GLM)-Gag 5 × 10⁶ NJ (GM)-Gag 5 × 10⁶ 6 7 Ind (GLM)-Gag 5 × 10⁷ NJ (GM)-Gag 5 × 10⁷ 6 8 Ind (GLM)-Gag 5 × 10⁸ NJ (GM)-Gag 5 × 10⁸ 6 9 Ind (GLM)-Gag 5 × 10⁹ NJ (GM)-Gag 5 × 10⁹ 6 10 NJ (WT)-Gag 5 × 10⁶ Ind (WT)-Gag 5 × 10⁶ 6 Total 60 Mice

TABLE 2  Neucleotide Sequence Comparison between M Genes of VSV Indiana serotype, Wild Type (SEQ ID NO: 1) and a Mutant G21E/L111F/M51R (SEQ ID NO 2)                1                                                   50 SEQ ID NO: 1:  ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC  SEQ ID NO: 2: ATGAGTTCCT TAAAGAAGAT TCTCGGTCTG AAGGGGAAAG GTAAGAAATC                 51                                                   100 SEQ ID NO: 1: TAAGAAATTA GGG ATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG  SEQ ID NO: 2: TAAGAAATTA GAA ATCGCAC CACCCCCTTA TGAAGAGGAC ACTAACATGG                 101                                                  150 SEQ ID NO: 1: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAG  SEQ ID NO: 2: AGTATGCTCC GAGCGCTCCA ATTGACAAAT CCTATTTTGG AGTTGACGAG                 151                                                  200 SEQ ID NO: 1: ATGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC  SEQ ID NO: 2: AGGGACACTC ATGATCCGCA TCAATTAAGA TATGAGAAAT TCTTCTTTAC                 201                                                  250 SEQ ID NO: 1: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG  SEQ ID NO: 2: AGTGAAAATG ACGGTTAGAT CTAATCGTCC GTTCAGAACA TACTCAGATG                 251                                                  300 SEQ ID NO: 1: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG  SEQ ID NO: 2: TGGCAGCCGC TGTATCCCAT TGGGATCACA TGTACATCGG AATGGCAGGG                 301                                                  350 SEQ ID NO: 1: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTG GGTTCTT CTAATCTAAA  SEQ ID NO: 2: AAACGTCCCT TCTACAAGAT CTTGGCTTTT TTT GGTTCTT CTAATCTAAA                 351                                                  400 SEQ ID NO: 1: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTC  SEQ ID NO: 2: GGCCACTCCA GCGGTATTGG CAGATCAAGG TCAACCAGAG TATCACGCTC                 401                                                  450 SEQ ID NO: 1: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCC  SEQ ID NO: 2: ACTGTGAAGG CAGGGCTTAT TTGCCACACA GAATGGGGAA GACCCCTCCC                 451                                                  500 SEQ ID NO: 1: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTA  SEQ ID NO: 2: ATGCTCAATG TACCAGAGCA CTTCAGAAGA CCATTCAATA TAGGTCTTTA                 501                                                  550 SEQ ID NO: 1: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGG  SEQ ID NO: 2: CAAGGGAACG GTTGAGCTCA CAATGACCAT CTACGATGAT GAGTCACTGG                 551                                                  600 SEQ ID NO: 1: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGAT  SEQ ID NO: 2: AAGCAGCTCC TATGATCTGG GATCATTTCA ATTCTTCCAA ATTTTCTGAT                 601                                                  650 SEQ ID NO: 1: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATC  SEQ ID NO: 2: TTCAGAGATA AGGCCTTAAT GTTTGGCCTG ATTGTCGAGA AAAAGGCATC                 651                                                  700 SEQ ID NO: 1: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGA  SEQ ID NO: 2: TGGAGCTTGG GTCCTGGATT CTGTCAGCCA CTTCAAATGA 

TABLE 3  Amino Acid Sequence Comparison between M Proteins of VSV Indiana serotype Wild Type(SEQ ID NO: 3) and a Mutant G21E/L111F/M51R (SEQ ID NO: 4)                1                    21                             50 SEQ ID NO: 3: MSSLKKILGL KGKGKKSKKL GIAPPPYEED TNMEYAPSAP IDKSYFGVDE SEQ ID NO: 4: MSSLKKILGL KGKGKKSKKL EIAPPPYEED TNMEYAPSAP IDKSYFGVDE                51                                                  100 SEQ ID NO: 3: MDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG  SEQ ID NO: 4: RDTHDPHQLR YEKFFFTVKM TVRSNRPFRT YSDVAAAVSH WDHMYIGMAG                 101       111                                       150 SEQ ID NO: 3: KRPFYKILAF LGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP SEQ ID NO: 4: KRPFYKILAF FGSSNLKATP AVLADQGQPE YHAHCEGRAY LPHRMGKTPP                151                                                  200 SEQ ID NO: 3: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD SEQ ID NO: 4: MLNVPEHFRR PFNIGLYKGT VELTMTIYDD ESLEAAPMIW DHFNSSKFSD                201                           229                    250 SEQ ID NO: 3: FRDKALMFGL IVEKKASGAW VLDSVSHFK SEQ ID NO: 4: FRDKALMFGL IVEKKASGAW VLDSVSHFK

TABLE 4  Nucleotide Sequence Comparison between M Genes of VSV New Jersey serotype Wild Type (SEQ ID NO: 5) and Mutants, G22E/M48R + M51R (SEQ ID NO: 6) and G22E/L110F/M48R + M51R (SEQ ID NO: 7) 1                                                    50 SEQ ID NO: 5: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA  SEQ ID NO: 6: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA  ID NO: 7: ATGAGTTCCT TCAAAAAGAT TCTGGGATTT TCTTCAAAAA GTCACAAGAA  51                                                  100 SEQ ID NO: 5: ATCAAAGAAA CTAGGC TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG  SEQ ID NO: 6: ATCAAAGAAA CTAGAA TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG  SEQ ID NO: 7: ATCAAAGAAA CTAGAA TTGC CACCTCCTTA TGAGGAATCA AGTCCTATGG  101                                                  150 SEQ ID NO: 5: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AATG GAGGAT  SEQ ID NO: 6: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AAGG GAGGAT  SEQ ID NO: 7: AGATTCAACC ATCTGCCCCA TTATCAAATG ACTTCTTCGG AAGG GAGGAT  151                                                  200 SEQ ID NO: 5: ATG GATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT  SEQ ID NO: 6: AGG GATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT  SEQ ID NO: 7: AGG GATTTAT ATGATAAGGA CTCCTTGAGA TATGAGAAGT TCCGCTTTAT  201                                                  250 SEQ ID NO: 5: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG  SEQ ID NO: 6: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG  SEQ ID NO: 7: GTTGAAGATG ACTGTTAGAG CTAACAAGCC CTTCAGATCG TATGATGATG  251                                                  300 SEQ ID NO: 5: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA  SEQ ID NO: 6: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA  SEQ ID NO: 7: TCACCGCAGC GGTATCACAA TGGGATAATT CATACATTGG AATGGTTGGA  301                                                  350 SEQ ID NO: 5: AAGCGTCCTT TCTACAAGAT AATTGCTCTG  ATTGGCTCCA GTCATCTGCA  SEQ ID NO: 6: AAGCGTCCTT TCTACAAGAT AATTGCTCTG  ATTGGCTCCA GTCATCTGCA  SEQ ID NO: 7: AAGCGTCCTT TCTACAAGAT AATTGCTTTT  ATTGGCTCCA GTCATCTGCA  351                                                  400 SEQ ID NO: 5: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA  SEQ ID NO: 6: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA  SEQ ID NO: 7: AGCAACTCCA GCTGTGTTGG CAGACTTAAA TCAACCAGAG TATTATGCCA  401 450 SEQ ID NO: 5: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG  SEQ ID NO: 6: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG  SEQ ID NO: 7: CACTAACAGG TCGTTGTTTT CTTCCTCACC GACTCGGATT GATCCCACCG  451                                                  500 SEQ ID NO: 5: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA  SEQ ID NO: 6: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA  SEQ ID NO: 7: ATGTTTAATG TGTCCGAAAC TTTCAGAAAA CCATTCAATA TTGGGATATA  501                                                  550 SEQ ID NO: 5: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG  SEQ ID NO: 6: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG  SEQ ID NO: 7: CAAAGGGACT CTCGACTTCA CCTTTACAGT TTCAGATGAT GAGTCTAATG  551                                                  600 SEQ ID NO: 5: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG  SEQ ID NO: 6: AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG  SEQ ID NO: 7 AAAAAGTCCC TCATGTTTGG GAATACATGA ACCCAAAATA TCAATCTCAG  601                                                  650 SEQ ID NO: 5: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC  SEQ ID NO: 6: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC  SEQ ID NO: 7: ATCCAAAAAG AAGGGCTTAA ATTCGGATTG ATTTTAAGCA AGAAAGCAAC  651                                                  700 SEQ ID NO: 5: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA  SEQ ID NO: 6: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA  SEQ ID NO: 7: GGGAACTTGG GTGTTAGACC AATTGAGTCC GTTTAA 

TABLE 5  Amino Acid Sequence Comparison between M Proteins of VSV New Jersey serotype Wild Type (SEQ ID NO: 8) and Mutants, G22E/M48R + M51R (SEQ ID NO: 9) and G22E/L110F/M48R + M51R (SEQ ID NO: 10) 1                      22                         48 50 SEQ ID NO: 8 : MSSFKKILGF SSKSHKKSKK LGLPPPYEES SPMEIQPSAP LSNDFFGMED SEQ ID NO: 9 : MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGRED SEQ ID NO: 10: MSSFKKILGF SSKSHKKSKK LELPPPYEES SPMEIQPSAP LSNDFFGRED 51                                                  100 SEQ ID NO: 8 : MDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG  SEQ ID NO: 9 : RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG  SEQ ID NO:10: RDLYDKDSLR YEKFRFMLKM TVRANKPFRS YDDVTAAVSQ WDNSYIGMVG  101     110                                         150 SEQ ID NO: 8: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP SEQ ID NO: 9: KRPFYKIIAL IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP SEQ ID NO: 10: KRPFYKIIAF IGSSHLQATP AVLADLNQPE YYATLTGRCF LPHRLGLIPP 151                                                  200 SEQ ID NO: 8: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ SEQ ID NO: 9: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ SEQ ID NO: 10: MFNVSETFRK PFNIGIYKGT LDFTFTVSDD ESNEKVPHVW EYMNPKYQSQ 201                                                 250 SEQ ID NO: 8: IQKEGLKFGL ILSKKATGTW VLDQLSPFK SEQ ID NO: 9: IQKEGLKFGL ILSKKATGTW VLDQLSPFK SEQ ID NO: 10: IQKEGLKFGL ILSKKATGTW VLDQLSPFK

TABLE 6 Vaccination studies in mice for broad range CD8+ T cell responses and humoral immune responses against HIV-1 proteins Vacci- nation rVSV w/HIV-1 Proteins Groups Prime Boost G 1 rVSV_(Ind)(GLM) rVSV_(NJ)(GM) G 2 rVSV_(Ind)(GLM)-HIV-1 Gag-En rVSV_(NJ)(GM)-HIV-1 Gag-En G 3 rVSV_(Ind)(GLM)-HIV-1 rVSV_(NJ)(GM)-HIV-1 Gp160mss Gp160mss G 4 rVSV_(Ind)(GLM)-HIV-1 Pol rVSV_(NJ)(GM)-HIV-1 Pol G 5 rVSV_(Ind)(GLM)-HIV-1 rVSV_(NJ)(GM)-HIV-1 Gag-En + rVSV_(Ind)(GLM)- Gag-En + rVSV_(NJ)(GM)- Gp160mss + rVSV_(Ind)(GLM)- Gp160mss + rVSV_(NJ)(GM)- HIV-1 Pol HIV1 Pol G 6 rVSV_(Ind)(GLM)-HIV-1 Gag-A + rVSV_(NJ)(GM)-HIV-1 Gag-A + rVSV_(Ind)(GLM)-HIV-1 Gag-B + rVSV_(NJ)(GM)-HIV-1 Gag-B + rVSV_(Ind)(GLM)-HIV-1 Gag-C rVSV_(NJ)(GM)-HIV-1 Gag-C 

What is claimed is:
 1. A prime boost combination vaccine, wherein the prime boost combination vaccine comprises: (a) an effective amount of a vaccine comprising an attenuated recombinant vesicular stomatitis virus (rVSV) of one serotype having a first modified M protein comprising the amino acid sequence of SEQ ID NO:4; and (b) an effective amount of a vaccine comprising a rVSV of another serotype having a second modified M protein comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
 2. The prime boost combination vaccine of claim 1, wherein (a) is a priming vaccine and (b) is a booster vaccine.
 3. The prime boost combination vaccine of claim 1, wherein (b) is a priming vaccine and (a) is a booster vaccine.
 4. The prime boost combination vaccine of claim 1, wherein the two attenuated rVSVs are chimeric rVSVs that express a protein of a foreign pathogen, and wherein the two chimeric rVSVs are capable of inducing an immune response to the protein.
 5. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is a viral, a fungal, a bacterial or a parasitic pathogen.
 6. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is a lentivirus.
 7. The prime boost combination vaccine of claim 6, wherein the lentivirus is a human immunodeficiency virus (HIV) and the protein is a HIV protein.
 8. The prime boost combination vaccine of claim 7, wherein the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HIV protein is inserted in between the G gene and the L gene.
 9. The prime boost combination vaccine of claim 8, wherein the HIV gene is selected from the group of HIV genes consisting of env, gag and pol.
 10. The prime boost combination vaccine of claim 4, wherein the foreign pathogen is hepatitis C virus (HCV) and the protein is a HCV protein.
 11. The prime boost combination vaccine of claim 10, wherein the rVSV of one serotype and the rVSV of the other serotype include a surface glycoprotein (G) gene and a RNA dependent RNA polymerase (L) gene, and wherein a gene for expressing the HCV protein is inserted in between the G gene and the L gene.
 12. The prime boost combination vaccine of claim 10, wherein the HCV protein is a structural or a non-structural HCV protein.
 13. The prime boost combination vaccine of claim 4, wherein each one of the two vaccines comprise a mixture of the attenuated chimeric rVSVs, and wherein two of the attenuated chimeric rVSVs in the mixture have a different protein of the foreign pathogen.
 14. The prime boost combination vaccine of claim 1, wherein each one of the two vaccines is capable of inducing humoral, cellular and mucosal immune responses.
 15. The prime boost combination vaccine of claim 1, wherein the serotype of vaccine (a) is Indiana and the serotype of vaccine (b) is New Jersey.
 16. The prime boost combination vaccine of claim 1, wherein each one of vaccine (a) and vaccine (b) further comprises an adjuvant. 